Determinants of SARS-CoV-2 entry and replication in airway mucosal tissue and susceptibility in smokers

. 2021 Sep 28;2(10):100421. doi: 10.1016/j.xcrm.2021.100421

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies

Rabbit polyclonal anti-ACE2 (Lot GR3333640)	Abcam	Cat# ab15348; RRID:AB_301861
Mouse monoclonal anti-TMPRSS2	Millipore Sigma	Cat# MABF2158
Mouse monoclonal anti-Acetylated α Tubulin	Santa Cruz	Cat# sc-23950; RRID:AB_628409
Rabbit monoclonal anti-Acetyl-α-Tubulin	Cell Signaling Technology	Cat# 5335; RRID:AB_10544694
Guinea pig polyclonal anti-Pan Cytokeratin	LSBio	Cat# LS-B16812
Mouse monoclonal anti-Tubulin β 3	Biolegend	Cat# MMS-435P; RRID:AB_2313773
Mouse monoclonal anti-CD45	Abcam	Cat# ab781; RRID:AB_306098
Mouse monoclonal anti-SARS-CoV-2 spike	GeneTex	Cat# GTX632604; RRID:AB_2864418
Donkey anti-Rabbit IgG (H+L) Highly Cross-Absorbed Secondary Antibody, Alexa Flour Plus 647	Thermo Fisher Scientific	Cat# A32795; RRID:AB_2762835
Donkey anti-Mouse IgG (H+L) Highly Cross-Absorbed Secondary Antibody, Alexa Flour Plus 555	Thermo Fisher Scientific	Cat# A32773; RRID:AB_2762848
Goat anti-Guinea Pig IgG (H+L) Highly Cross-Absorbed Secondary Antibody, Alexa Flour 488	Thermo Fisher Scientific	Cat# A11073; RRID:AB_2534117
Phalloidin-Atto 647N	Millipore Sigma	Cat# 65906

Bacterial and virus strains

SARS-CoV-2	Joe DeRisi lab, UCSF	SARS-CoV-2/human/USA/CA-UCSF-0001C/2020

Biological samples

Human nasal, nasopharynx, oro/hypo-pharynx, larynx, bronchus, uvula, tonsil, and conjunctiva tissues	Stanford University School of Medicine	N/A
Human Tissue Microarray	US Biolab	Cat# XHN801
Human Tissue Microarray	US Biolab	Cat# XHN803
Human Paraffin Embedded Tissue Array	US Biomax	Cat# OR208a
Human Paraffin Embedded Tissue Array	US Biomax	Cat# BN1002b
Human Paraffin Embedded Tissue Array	US Biomax	Cat# LP208
Human nasal epithelial cells	Stanford University School of Medicine	N/A
MucilAir	Epithelix	Cat# EP01MD

Chemicals, peptides, and recombinant proteins

The ACE2 peptide for blocking assay: KGENNPGFQNTDDVQTSF	GenScript	N/A
The TMPRSS2 peptide for blocking assay: (sequence provided in confidence by the manufacturer)	GenScript	N/A
RNAscope Hydrogen Peroxide Reagent	Bio-Techne	Cat# 322335
Dako Target Retrieval Solution, pH9	Dako Agilent	Cat# S236784-2
Recombinant Human Interferon-β1	Peprotech	Cat# 300-02BC
RNeasy Lipid Tissue Kit	QIAGEN	Cat# 74804
M-MLV Reverse Transcriptase	Invitrogen	Cat# 28025-013
TaqMan Gene Expression Master Mix	Applied Biosystems	Cat# 4369016

Critical commercial assays

RNAscope Multiplex Fluorescent Reagent Kit v2	Bio-Techne	Cat# 323100
TSA Cyanine 3	Akoya Biosciences	Cat# NEL744001KT
TSA Cyanine 5	Akoya Biosciences	Cat# NEL745001KT

Deposited data

Single cell RNA-seq data³⁰	NCBI Gene Expression Omnibus	GEO: GSE134174
Single cell RNA-seq data²⁸	European Genome-Phenome Archive	EGAD00001006185
Single cell RNA-seq data²⁹	NCBI Gene Expression Omnibus	GEO: GSE103322
Single cell RNA-seq data²⁵	European Genome-Phenome Archive	EGAS00001001755
Single cell RNA-seq data²⁶	Single Cell Portal	https://singlecell.broadinstitute.org/single_cell/study/SCP253/allergic-inflammatory-memory-in-human-respiratory-epithelial-progenitor-cells#study-download

Oligonucleotides

SARS-CoV-2 Spike mRNA	Bio-Techne	Cat# 848561
IFNB1 mRNA	Bio-Techne	Cat# 417071
IL6 mRNA	Bio-Techne	Cat# 310371
ACE2 (Hs00222343_m1)	Life Technologies	Cat# 4331182
TMPRSS2 (Hs00237175_m1)	Life Technologies	Cat# 4331182
IFNB1 (Hs01077958_s1)	Life Technologies	Cat# 4331182

Software and algorithms

GraphPad Prism 6	GraphPad	https://www.graphpad.com/
IBM SPSS Statistics version 23	IBM	https://www.ibm.com/support/pages/downloading-ibm-spss-statistics-23
Seurat v4.0	Seurat³¹	https://satijalab.org/seurat/
Single R	Aran et al.⁶⁸	https://github.com/dviraran/SingleR
ImageJ v1.52t	NIH	https://imagej.net/downloads
deepcell-tf 0.6.0	Greenwald et al.⁶⁹; Van Valen et al.⁷⁰	https://github.com/vanvalenlab/deepcell-tf